Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance

Abstract

The balance between germ-line stem cell (GSC) self-renewal and differentiation in Drosophila ovaries is mediated by the antagonistic relationship between the Nanos (Nos)-Pumilio translational repressor complex, which promotes GSC self-renewal, and expression of Bam, a key differentiation factor. Here, we find that Bam and Nos proteins are expressed in reciprocal patterns in young germ cells. Repression of Nos in Bam-expressing cells depends on sequences in the nos 3'-UTR, suggesting that Nos is regulated by translational repression. Ectopic Bam causes differentiation of GSCs, and this activity depends on the endogenous nos 3'-UTR sequence. Previous evidence showed that Bgcn is an obligate factor for the ability of Bam to drive differentiation, and we now report that Bam forms a complex with Bgcn, a protein related to the RNA-interacting DExH-box polypeptides. Together, these observations suggest that Bam-Bgcn act together to antagonize Nos expression; thus, derepressing cystoblast-promoting factors. These findings emphasize the importance of translational repression in balancing stem cell self-renewal and differentiation.

Fig. 1.

Bam and Nos are expressed in reciprocal patterns. (A) Bam protein (red, detected by anti-GFP against a Bam:GFP rescuing transgene) and Nos protein (green, detected by anti-myc against a Nos:Myc rescuing transgene) are expressed in nonoverlapping domains in early germ cells. Stem stems (arrows), CBs (arrowheads). (B and C) Germ cells from encore mutant flies (C) display an expansion of Bam expression compared with enc/+ and (B) a corresponding delay in late Nos accumulation (red, anti-Bam; green, Nos:GFP, detected by anti-GFP against a rescuing transgene). Stem cells (arrows), CBs (arrowheads). Cystoblasts are out of plane of focus in C. (D) Nos (green) remains high in germ cells lacking Bam. (A′–D′) Nos channel alone. (Magnification bars, 25 μm.)

Fig. 2.

Genetic interactions reveal an antagonistic relationship between bam and nos. (A) Ovaries from nos[RC] mutants are rudimentary (arrow) with few maturing egg chambers (arrowhead). (B) Introduction of a single loss of function bam allele suppressed the nos[RC] phenotype and ovaries contained many more maturing egg chambers (arrows). (C) Suppression was quantified by counting the number of egg chambers per terminal filament in ovaries from nos[RC] and nos[RD] ± bam or bgcn. The difference between nos[RC]/Df and nos[RD]/Df may reflect suppressing background mutation(s) on the nos[RD] chromosome, because the bam[BG] nos[RD] recombinant chromosome produced a more severe phenotype over Df than the original nos[RD] chromosome.

Fig. 3.

Nos accumulation depends on the 3′-UTR. (A) Nos promoter driving GFP expression accumulates GFP throughout region 1 of the germarium. Stem cells (arrows), CBs (arrowheads). (B) Nos protein (green, anti-GFP against a rescuing nos-GFP transgene) accumulates in stem cells (arrow) and precystoblasts, is lost in Bam+ cells (red, anti-Bam), and reaccumulates in 16-cell cysts where Bam expression is no longer detected. (C) Nos expressed from the nos promoter but with the heterologous tubulin 3′-UTR accumulates uniformly in region 1 of the germarium, including in Bam+ cells (arrowhead). (A′–C′) Nos channel alone.

Fig. 4.

Bam-dependent GSC loss acts via the nos 3′-UTR. (A–C) Green, Vasa; Red, Hts; and Blue, DNA. (A) The p[HS-bam] ovaries maintained at 37 °C for 1 h were examined 3 days post heat shock; 86% of these germaria were devoid of stem cells. (B) Germaria from animals carrying a wild-type nos transgene were similarly depleted of stem cells by HS-bam treatment (71% of germaria had no stem cells). Arrow points to a 4-cell cyst and arrowhead indicates a CB or 2-cell cyst in the GSC position. (C) Germaria from animals carrying a nos transgene with a tubulin 3′-UTR were protected from HS-bam induced stem cell depletion. Arrows indicate 2 GSCs (81% of germaria contained 1 or more stem cells). (D) Quantification of GSC retention or loss after ectopic Bam expression. (E–H) Green, Nos:GFP; Red, Hts; and Blue, DNA. Arrows point to GSCs. Detectible Nos declines in stem cells after HS-bam, except when Nos is expressed with the tubulin 3′-UTR. (E) Nos:GFP expression levels in germarium of p[nosP-Nos:GFP-nos3′-UTR] animal without heat shock. (F) Nos:GFP levels in p[nosP-Nos:GFP-nos3′-UTR] animal 9.5 h after heat shock. Arrows indicate GSCs; arrowheads indicate 16-cell cysts. (G) Nos:GFP expression levels in germarium of p[nosP-Nos:GFP-tub3′-UTR] animal without heat shock. (H) Nos:GFP expression levels in germarium of p[nosP-Nos:GFP-tub3′-UTR] animal 9.5 h after heat shock. Arrows indicate GSCs; arrowheads indicate 16-cell cysts.

Fig. 5.

Bam and Bgcn form a complex. (A) Immunoblot probed with anti-V5 antibody after “pulldown” experiments with epitope-tagged Bam:GST and Bgcn:V5 proteins. (lane 1) Yeast extract expressing Bgcn:V5. (lanes 2–4) Samples from pellets recovered by incubation with glutathione-coupled agarose beads (GST pulldowns). Bgcn:V5 was recovered in the pellet only when the extracts from Bgcn:V5 yeast were incubated with Bam:GST bacterial extracts. (B) Bam and Bgcn coimmunoprecipitate from ovaries. Ovarian extracts from flies expressing Bgcn:GFP and Bam:HA (lane 2) or w¹¹¹⁸ flies (lane 3) were incubated with anti-GFP or anti- HA antibodies for immunoprecipitation and the immunoblots were probed with anti-GFP and anti-HA antibodies separately. Lane 1, 10% of extract; lane 2, flies w⁻;[w⁺,bamP-Bam:HA];[w⁺,bgcnP-Bgcn:GFP] anti-GFP IP; lane 3, flies w¹¹¹⁸ anti-GFP IP; lane 4, flies w⁻;[w⁺,bamP-Bam:HA];[w⁺,bgcnP-Bgcn:GFP] anti-HA IP; lane 5, flies w¹¹¹⁸ anti-HA IP. (C) Bam and Bgcn coimmunoprecipitate from S2 cells that expressed tagged proteins. Tagged proteins (Bam:HA; Bgcn:Myc) were over expressed in S2 cells and the cell lysate were used for anti-HA or anti-Myc immunoprecipitation. Lane 1, 10% of S2 cell lysate; lane 2, S2 cell lysate expressing Bam:HA and Bgcn:Myc immunoprecipitated with anti-HA antibody; lane 3, S2 cell lysate expressing Bgcn:Myc immunoprecipitated with anti-HA antibody; lane 4, S2 cell lysate expressing Bam:HA and Bgcn:Myc immunoprecipitated with anti-Myc antibody; lane 5, S2 cell lysate expressing Bam:HA immunoprecipitated with anti-Myc antibody.